SYBR Green I, a commonly used fluorescent DNA binding dye, binds all
double–stranded DNA and detection is monitored by measuring the
increase in fluorescence throughout the cycle. SYBR Green I has an
excitation and emission maxima of 494 nm and 521 nm, respectively.
Specificity of Sigma's SYBR based QPCR detection is greatly enhanced by
the incorporation of a hot–start mediated taq polymerase, JumpStart Taq.

Convenient – Delivers the benefits of antibody–inactivated hot–start
PCR with SYBR Green detection in a ReadyMix ideal for high throughput
applications; only primers and template are required.
Specific – JumpStart Taq antibody prevents non–specific product
formation through its hot–start mechanism.
Flexible – SYBR Green JumpStart Taq ReadyMixes for SYBR based QPCR are
formulated with MgCl2 or packaged with a separate vial for ease of
optimization. Additionally, our ReadyMixes are compatible with tube-
and plate- based instruments.

Probe based
Quantitative PCR

Probe based QPCR relies on the sequence–specific detection of a desired
PCR product. Unlike SYBR based QPCR methods that detect all
double–stranded DNA, probe based QPCR utilizes a fluorescent–labeled
target-specific probe resulting in increased specificity and
sensitivity. Additionally, a variety of fluorescent dyes are available
so that multiple primers can be used to simultaneously amplify many
sequences.

Ideal for high throughput. ReadyMixes contain all necessary components
for QPCR you simply add the fluorescent detection chemistry, primers,
and template.
Specific – JumpStart Taq antibody prevents non–specific product
formation through it hot–start mechanism.
Maximum flexibility in detection method, since no detection method has
been incorporated into formulation.
Optimized formulations with the addition of dUTP to facilitate
carry–over prevention from previous PCR.

Real-time
PCR is the method of choice in many laboratories for
diagnostic and food applications. This technology merges the polymerase
chain reaction chemistry with the use of fluorescent reporter molecules
in order to monitor the production of amplification products during
each cycle of the PCR reaction. Thus, the combination of excellent
sensitivity and specificity, reproducible data, low contamination risk
and reduced hand-on time, which make it a post-PCR analysis
unnecessary, has made real-time PCR technology an appealing alternative
to conventional PCR. The present paper attempts to provide a rigorous
overview of fluorescent-based methods for nucleic acid analysis in
real-time PCR described in the literature so far. Herein, different
real-time PCR chemistries have been classified into two main groups;
the first group comprises double-stranded DNA intercalating molecules,
such as SYBR Green I and EvaGreen, whereas the second includes
fluorophore-labeled oligonucleotides. The latter, in turn, has been
divided into three subgroups according to the type of fluorescent
molecules used in the PCR reaction: (i) primer-probes (Scorpions,
Amplifluor, LUX, Cyclicons, Angler); (ii) probes; hydrolysis (TaqMan,
MGB-TaqMan, Snake assay) and hybridization (Hybprobe or FRET, Molecular
Beacons, HyBeacon, MGB-Pleiades, MGB-Eclipse, ResonSense, Yin-Yang or
displacing); and (iii) analogues of nucleic acids (PNA, LNA, ZNA,
non-natural bases: Plexor primer, Tiny-Molecular Beacon). In addition,
structures, mechanisms of action, advantages and applications of such
real-time PCR probes and analogues are depicted in this review.

SYBR Green I is
dsDNA-binding dye. It is thought to bind in the minor groove of
dsDNA and
upon binding increases in fluorescence over a hundred fold (Figure
8a). It is compatible with PCR up to a point, at very high
concentrations
it starts to inhibit the PCR reaction. In the LightCycler
Instrument, SYBR is monitored in channel F1. The biggest advantage of
SYBR is that
it binds to any dsDNA; there is no designing and optimizing of probes
required. If you have a PCR that works, you can have a real-time
quantitative assay working in about a day. The biggest
disadvantage of SYBR is that it binds to any dsDNA; the specific
product, non-specific products and primer dimers are detected equally
well. There are a number of ways to handle this problem.
Careful optimization of the PCR reaction can usually reduce primer
dimers to a level that is only important for very low copy
detection. Hot start techniques like TaqStart antibody can be
helpful in reducing primer dimer. The LightCycler Instrument
allows melting curve analysis of the reaction. This can help to
determine the fraction of the signal coming from the desired product
and the fraction coming from primer dimer. Once the
melting point of the product has been determined the LightCycler
Instrument's flexible programming allows the user to acquire
fluorescence above the melting
temperature of the primer dimers, but below the melting temperature of
the product.

Hybridization
Probes

If sequence
specific recognition is required, the HybProbe system allows detection
of only the specific product. Two probes are designed that
hybridize side by side on the PCR product (Figure 8c). The 3’ end
of the upstream probe is labeled with fluorescein, which acts as a
fluorescence resonance energy transfer (FRET) donor. The 5’ end
of the downstream probe is labeled with an acceptor dye, either LC Red
640, or LC Red 705. The FRET signal is seen only when two
specific hybridization events occur. In the LightCycler
Instrument, LC Red 640 is monitored in channel F2, LC Red 705 in
channel F3. There may sometimes be an advantage to monitoring the
ration of the acceptor channel (where the signal goes up with
increasing PCR product) and the signal from fluorescein in F1 (which
goes down with increasing PCR product.

TaqMan®
Probes

TaqMan probes
derive their fluorescence signal from the hydrolysis of the probe by
Taq’s 5’ to 3’ exonuclease activity (Figure 8c). The hydrolysis
separates fluorescein from a quenching dye and results in an increased
fluorescein signal. These probes can be used in the LightCycler
Instrument and are monitored in F1 or F1/F2.

The fluorescent
dye SYBR Green I binds to the minor groove of the DNA double helix. In
solution, the unbound dye exhibits very little fluorescence, however,
fluorescence is greatly enhanced upon DNA-binding. Since SYBR Green I
dye is very
stable (only 6% of the activity is lost during 30 amplification cycles)
and the LightCycler instrument's optical filter set matches the
wavelengths of excitation and emission, it is the reagent of choice
when measuring
total DNA. The principle is outlined in the following figures.

At the beginning
of amplification, the reaction mixture contains the denatured DNA, the
primers, and the dye. The unbound dye molecules weakly fluoresce,
producing a minimal background fluorescence signal which is subtracted
during computer analysis.

After annealing
of the
primers, a few dye molecules can bind to the double strand. DNA binding
results
in a dramatic increase of the SYBR Green I molecules to emit light upon
excitation.

During
elongation, more
and more dye molecules bind to the newly synthesized DNA. If the
reaction is monitored continuously, an increase in fluorescence is
viewed in real-time. Upon denaturation of the DNA for the next heating
cycle, the
dye molecules are released and the fluorescence signal falls.

Fluorescence
measurement at the end of the elongation step of every PCR cycle is
performed
to monitor the increasing amount of amplified DNA. Together with a
melting curve analysis performed subsequently to the PCR, the SYBR
Green
I format provides an excellent tool for specific product identification
and quantification.

SYBR Green I (SG) is widely used
in real-time PCR applications as an intercalating dye and is included in many commercially available
kits at undisclosed concentrations. Binding of SG to double-stranded DNA is non-speciÆc and
additional testing, such as DNA melting curve analysis, is required to
conÆrm the generation of a speciÆc
amplicon. The use of melt curve analysis eliminates the necessity for agarose gel electrophoresis
because the melting temperature (Tm) of the speciÆc amplicon is analogous to the detection of an
electrophoretic band. When using SG for real-time PCR multiplex reactions, discrimination of
amplicons should be possible, provided the Tm values are suffiently different. Real-time multiplex
assays for Vibrio cholerae and
Legionella pneumophila using commercially available kits and in-house SG
mastermixes have highlighted variability in performance characteristics, in particular the
detection of only a single
product as assessed by Tm analysis but multiple products as assessed by
agarose gel electrophoresis.
The detected Tm corresponds to the amplicon with the higher G+C%
and larger size, suggesting
preferential binding of SG during PCR and resulting in the failure to
detect multiple amplicons in multiplex reactions when the amount of SG present is limiting. This
has implications for the design and routine application of diagnostic real-time PCR assays employing SG.

The minor groove binding
asymmetric cyanine dye 4-[(3-methyl-6- (benzothiazol-2-yl)-
2,3-dihydro- (benzo-1,3-thiazole)
-2-methylidene)]- 1-methyl-pyridinium iodide (BEBO) is tested as
sequence nonspeciÆc label in real-time PCR. The
Fluorescence intensity of BEBO increases upon binding to double-stranded DNA allowing
emission to be measured at the end of the elongation phase in the PCR cycle. BEBO concentrations
between 0.1 and 0.4 mM generated sufÆcient Øuorescence
signal without
inhibiting the PCR. A comparison with the commonly used reporter dye
SYBR Green I shows that the two dyes behave similarly
in all important aspects.

BEBO
for qPCR and HRM

TATAA Biocenter
AB, Göteborg, Sweden

BEBO is
an unsymmetric cyanine dye developed by TATAA Biocenter for use in qPCR
applications.

The dye has absorbance and emission wavelengths that can be detected on
the FAM channel on most common real-time PCR platforms, and shows a
strong fluorescence increase when bound to dsDNA. BEBO can be used as
an unspecific dye for real-time PCR applications or other applications
where staining of dsDNA is wanted.

Currently,
in real-time PCR, one often has to choose between using asequence-specific
probe and a nonspecific double-stranded DNA (dsDNA) binding dye
for the detection of amplified DNA products. The sequence-specific
probe has the advantage that it
only detects the
targeted product, while the nonspecific dye has the
advantage that melting curve analysis can be performed after completed
amplification, which reveals what kind of
products have been formed. Here we present a new
strategy based on combining a sequence-specific probe and a
nonspecific dye, BOXTO, in the same reaction, to take the advantage of
both chemistries.
We show that BOXTO can be used
together with both TaqMan probes and locked nucleic
acid (LNA) probes without interfering with the PCR. The probe signal
reflect formation of target product, while melting curve analysis of
the BOXTO
signal reveals primer-dimer formation and the
presence of any other anomalous products.

The
unsymmetrical cyanine dyes BOXTO
(4-[6-(benzoxazole-2-yl-(3-methyl-)-2,3-dihydro-
(benzo-1,3-thiazole)-2-methylidene)]- 1-methyl-quinolinium chloride)
and its positive divalent derivative BOXTO-PRO
(4-[3-methyl-6-(benzoxazole-2-yl)-
2,3-dihydro- (benzo-1,3-thiazole)-2-methylidene)]-
1-(3-trimethylammonium-propyl)- quinolinium dibromide) were studied as
real-time PCR reporting fluorescent dyes and compared to SYBR GREEN I
(SG) (2-[N-(3-dimethylaminopropyl)-N-propylamino]-
4-[2,3-dihydro-3-methyl- (benzo-1,3-thiazol-2-yl)-methylidene]-
1-phenylquinolinium). Unmodified BOXTO showed no inhibitory effects on
real-time PCR, while BOXTO-PRO showed complete inhibition, Sufficient
fluorescent signal was acquired when 0.5–1.0 µM BOXTO was used
with
RotorGene and iCycler platforms. Statistical analysis showed that there
is no significant difference between the efficiency and dynamic range
of BOXTO and SG. BOXTO stock solution (1.5 mM) was stable at −20°C
for
more than one year and 40 µM BOXTO solution was more stable than
5x SG
when both were stored at 4°C for 45 days.

Real-time
systems for PCR were improved by probe-based, rather than
intercalator-based, PCR product detection. The principal drawback to
intercalator-based detection of PCR product accumulation is that both
specific and nonspecific products generate signal. An alternative
method, the 5' nuclease assay, provides a real-time method for
detecting only specific amplification products. During amplification,
annealing of the probe to its target sequence generates a substrate
that is cleaved by the 5' nuclease activity of Taq DNA polymerase when
the enzyme extends from an upstream primer into the region of the
probe. This dependence on polymerization ensures that cleavage of the
probe occurs only if the target sequence is being amplified.The development of fluorogenic probes made it possible to
eliminate post-PCR processing for the analysis of probe degradation.
The probe is an oligonucleotide with both a reporter fluorescent dye
and a quencher dye attached. While the probe is intact, the proximity
of the quencher greatly reduces the fluorescence emitted by the
reporter dye by Förster resonance energy transfer (FRET) through
space. Probe design and synthesis has been simplified by the finding
that adequate quenching is observed for probes with the reporter at the
5' end and the quencher at the 3' end.Figure 1 diagrams what happens to a fluorogenic probe
during the extension phase of PCR. If the target sequence is present,
the probe anneals downstream from one of the primer sites and is
cleaved by the 5' nuclease activity of Taq DNA polymerase as this
primer is extended. This cleavage of the probe separates the reporter
dye from quencher dye, increasing the reporter dye signal. Cleavage
removes the probe from the target strand, allowing primer extension to
continue to the end of the template strand. Thus, inclusion of the
probe does not inhibit the overall PCR process. Additional reporter dye
molecules are cleaved from their respective probes with each cycle,
effecting an increase in fluorescence intensity proportional to the
amount of amplicon produced.The advantage of fluorogenic probes over DNA binding dyes
is that specific hybridization between probe and target is required to
generate fluorescent signal. Thus, with fluorogenic probes,
non-specific amplification due to mis-priming or primer-dimer artifact
does not generate signal. Another advantage of fluorogenic probes is
that they can be labeled with different, distinguishable reporter dyes.
By using probes labeled with different reporters, amplification of two
distinct sequences can be detected in a single PCR reaction. The
disadvantage of fluorogenic probes is that different probes must be
synthesized to detect different sequences:

LightCycler
Hybridisation Probes

The detection
principle of LC™ Hybridization Probes (HybProbes) is Fluorescence
Resonance Energy Transfer (FRET), the phenomenon of energy transfer
from a donor to an acceptor fluorophor. If the donor and the acceptor
fluorophor are in close proximity to each other, excitation of the
donor by blue light results in energy transfer to the acceptor, which
can then emit light of longer wavelength. This fact forms the basis for
Roche’s real-time online LightCycler™ PCR System. It allows formation
of PCR products to be monitored by using two sequence specific,
fluorescent labeled oligonucleotide probes, called Hybridization
Probes, in addition to the PCR primers.

For this LC™
real-time PCR detection format the following are the major steps:

a)

DenaturationPCR template,
primers and HybProbes are single-stranded. One HybProbe is labeled with
the fluorescent donor dye Fluorescein, the other one is labeled with
one of the two available acceptor dyes (LCRed 640 or LCRed 705). The
donor dye is excited by blue light of 470 nm and emits green light
of 530 nm.

b)

AnnealingAfter reaching
the annealing temperature, PCR primers and HybProbes hybridise to their
specific target regions. The donor dye now comes into close proximity
to the acceptor dye. Energy emitted from the donor dye excites the
acceptor dye, which now emits red light of 640 or 705 nm.

c)

ElongationAfter
annealing to their target sites, the primers are elongated by
thermostable DNA polymerase. Due to the increased temperature of
72°C during elongation, most HybProbes have already melted off.
Probes that are still annealed to their target sequence are displaced
by the protruding DNA polymerase.

d)

CompletionThe amount of
template DNA has doubled and the DNA is double-stranded HybProbes are
displaced from their target site. The next cycle of PCR is ready
to start again at step a).

HybProbes are
designed as a pair of which one probe is labeled with the donor
(3´Fluo) and one with the acceptor (5´ LCRed 640 or LCRed
705) dye. As FRET decreases with the sixth power of distance,
HybProbes have to be designed to hybridise to adjacent regions of the
template DNA (separated by 1-5 nucleotides). If both probes hybridise,
the two dyes are brought close together and FRET to the
acceptor dye results in a signal measurable by the built-in fluorimeter
of the LightCycler™.

The fluorescence
signal disappears by increasing temperature above the melting
temperature of the oligos because the probes melt away from the
template strand which significantly increases the distance between the
dyes.

Mismatches
between the
probes and the target decrease the melting temperature of the
respective probe
compared to a perfectly matched probe. This effect can also be used to
detect
SNPs by melting curve analysis.

The Hybridization
Probe format is used for DNA detection and quantification and provides
a maximal specificity for product identification. In addition to the
reaction components used for conventional PCR, two specially designed,
sequence specific oligonucleotides labeled with fluorescent dyes are
applied
for this detection method. This allows highly specific detection of the
amplification product as described below.

The top
figure shows the three essential components for using
fluorescence-labeled oligonucleotides as Hybridization Probes: two
different oligonucleotides (labeled) and the amplification product.
Oligo 1 carries a fluorescein label at its 3' end whereas oligo 2
carries another label (LC Red 640) at
its 5' end.

The
sequences of the two oligonucleotides are selected such that they
hybridize to the amplified DNA fragment in a head to tail arrangement.
Why is this design important? When the oligonucleotides hybridize in
this orientation, the two fluorescence dyes are positioned in close
proximity to each other.

The first dye
(fluorescein) is excited by the LightCycler's LED (Light Emitting
Diode) filtered light source, and emits green fluorescent light at a
slightly longer wavelength (middle figure). When the two dyes are in
close proximity (as shown in the lower figure), the emitted energy
excites the LC Red
640 attached to the second hybridization probe that subsequently emits
red fluorescent light at an even longer wavelength. This energy
transfer,
referred to as FRET (Fluorescence Resonance Energy Transfer) is highly
dependent on the spacing between the two dye molecules. Only if the
molecules
are in close proximity (a distance between 1–5 nucleotides) is the
energy
transferred at high efficiency. Choosing the appropriate detection
channel,
the intensity of the light emitted by the LightCycler – Red 640 is
filtered
and measured by the LightCycler instrument's optics.

The increasing
amount of measured fluorescence is proportional to the increasing
amount of DNA generated during the ongoing PCR process. Since LC Red
640 only emits a signal when both oligonucleotides are hybridized, the
fluorescence measurement is performed after the annealing step.
Hybridization probes can be labeled with LightCycler – Red 640 and with
LightCycler – Red 705.

The
most difficult qPCR applications demand double-quenched probes for
optimum performance. BHQnova™ probes
have improved quenching efficiency compared to traditional end-labeled
probes, while also improving signal release upon amplification. BHQnova
is most advantageous in longer probe designs, typically those over 25
bases, to boost the signal-to-noise ratio by overcoming the upper limit
on sequence length.

The unique
combination of online available assay design software and
only 165 prevalidated, real-time PCR probes allows to quantify
virtually any transcript in the transcriptomes of a large number of
organisms. Universal
ProbeLibrary probes are fully compatible with commonly used
PCR conditions and the hydrolysis probe detection format. They are
labeled at the 5' end with fluorescein (FAM) and at the 3' end with a
dark quencher dye.

Flexibility,
specificity, convenience - all in one with the Universal ProbeLibraryThe
Universal ProbeLibrary combines the flexibility, availability and
covenience of SYBR Green I assays with the specificity of Hydrolysis
Probe assays. Just 165 prevalidated
probes, that can easily be stored in your freezer are sufficient to
quantify virtually any transcript from the transcriptomes of a large
number of organisms. Target specific
intron-spanning qPCR assays are designed online with
the ProbeFinder software, freely available at the Universal
ProbeLibrary Assay Design Center. The complete assay information,
including the sequence of specific primer pairs, and the appropriate
Universal ProbeLibrary probe, probe location, amplification product, is
displayed on the result page.

Molecular Beacons are oligonucleotide probes that emit fluorescence
when hybridised to
a target sequence of DNA or RNA.
These probes undergo a conformational change when they hybridise to
their target. The
stem and loop structure is made up by a loop structure which is a
complementary
sequence to the target sequence being detected, and the stem is formed
by the annealing
of complementary arm sequences that are on the end of the probe
sequence.

On the end of one arm, a fluorescent moiety is covalently attached,
whilst at the end of the
other arm is a quenching moiety also covalently attached. Due to the
stem structure both
moieties are kept in close proximity and the fluorescence is quenched
by energy transfer.
When the probe encounters it's target sequence a probe-hybrid is
formed, which is longer
and more stable than the stem-hybrid.
This conformational change forces the arm sequences apart, leading
to an increase in
fluorescence.

Differently-colored
molecular probes specific for the wild-type and mutant alleles are
designed. DNA amplified from homozygous wild-type individuals binds
only to the fluorescein-labeled molecular beacons (left). DNA from
homozygous mutants binds only the tetramethylrhodamine-labeled
molecular beacons (right). Both types of molecular probes will bind to
amplicons generated from the DNA of heterozygous individuals
(center).

Our genotyping
process is based on Scorpions Technology - a homogeneous or closed tube
method with a simple mix and glow operation. A DNA sample is added to a
Scorpions test and an increase in fluorescence indicates the genotype.
There is no post-PCR manipulation and the use of two fluorescent dyes
gives single tube SNP analysis

Scorpions is a
class leading PCR detection technology with significant benefits over comparable
approaches. These include:

stronger signals

lower backgrounds

faster reactions

simplified design

improved SNP tests

extended multiplexes

ideal for sample pooling

straightforward manufacture

How Scorpions Works

Scorpions
are bi-functional molecules containing a PCR
primer element covalently linked to a probe
element. The molecules also contain a fluorophore that
can interact with a quencher to reduce fluorescence. When the molecules are
used in a PCR reaction the fluorophore and the quencher are separated which
leads to an increase in light output from the reaction tube.

The
benefits
of Scorpions derive from the fact that the probe element is physically
coupled
to the primer element - this means that the reaction leading to signal
generation
is a uni-molecular rearrangement. This contrasts to the bi-molecular
collisions
required by other technologies such as Taqman or Molecular Beacons.

The benefits of a
uni-molecular rearrangement are significant - as the reaction is
effectively instantaneous it occurs prior to any competing or side
reactions such as target
amplicon re-annealing or inappropriate target folding. This leads
to stronger signals, more reliable probe design, shorter reaction times
and better discrimination.

Scorpions

The presence of
the blocker group is an essential element of the Scorpions invention.
Without such a blocker the Taq DNA polymerase would be able to read
through the Scorpions primer and copy the probe region. This would
generate signal but not in a target specific fashion. Copying the tail
in this way would completely negate the benefits of the Scorpions
reaction as any inappropriate side-reactions, including the formation
of primer dimers, would also generate a signal.

Scorpions are PCR
primers with a " Stem-Loop " tail containing a fluorophore and a
quencher
(figue1).The Stem-Loop tail
is separated from the PCR primer sequence by a " PCR stopper ", a
chemical modification that prevents the PCR from copying the stem-loop
sequence of the Scorpions primer. During PCR, the Scorpions primers are
extended to form PCR products. At the appropriate stage in the PCR
cycle (the annealing phase), the probe sequence in the Scorpion tail
curls
back to hybridize to the target sequence in the PCR product (figure 2).
As the tail of the scorpion and the PCR product are now part of the
same
strand of DNA, the interaction is intermolecular. The target sequence
is
typically chosen to be within 3 bases of the 3'end of the Scorpion
primer.
A Scorpion
consists of a specific probe sequence that is held in a hairpin loop
configuration by complementary stem sequence on either end. A
fluorophore is attached to the 5' end giving a fluorescent signal that
is quenched in the hairpin loop configuration by a moeity joined to the
3'end. The haipin loop is linked to the 5' end of a primer.After extension of
the Scorpion primer, during amplification, the
specific probe sequence is able to bind to its complement within the
same strand of DNA. This hybridization event opens the hairpin
loop so that fluorescence is not longer quenched and an increase in
signal is observed. A PCR stopper between the primer and the stem
sequence
prevents read-though of the hairpin loop, which could lead to the
opening
of the hairpin loop in the absence of the specific target sequence. The
unimolecular nature of the hybridization event gives rise to
significant
advantages over homogeneous probe systems. Unlike Molecular
Beacon
and Double-Dye Oligonucleotides assays (for which Scorpions can be used
as an aternative technology), Scorpion assays do not require a separate
probe.

Molecular
diagnostics is progressing from low-throughput, heterogeneous, mostly
manual technologiesto higher throughput, closed-tube, and automated methods.
Fluorescence is the favored signaling technology for such assays,
and a
number of techniques rely on energy transfer between a fluorophoreand a proximal
quencher molecule. In these methods, dual-labeled probes hybridize
to an amplicon andchanges in the quenching of
the fluorophore are detected. We describe a new technology that is
simple touse,
gives highly specific information, and avoids the major difficulties of
the alternative methods. It usesa primer with an integral tail
that is used to probe an extension product of the primer. The probing
of a targetsequence
is thereby converted into a unimolecular event, which has substantial
benefits in terms ofkinetics, thermodynamics, assay design, and probe
reliability.

The
effects of comprehensive LNA substitution in PCR primers for
amplification of human genomic DNA targets are presented in this
report. Previous research with LNA in other applications has shown
interesting properties for molecular hybridization including enhanced
specificity in allele-specific PCR.
Here we systematically modified PCR primers and conditions for the
human
genomic DNA targets APOB and PAH, along with a b-globin amplification
control, to study whether the number and position of LNA residues
improves
or diminishes amplification sensitivity and specificity. It was
observed
that the design rules for LNA substitution in PCR primers are complex
and depend upon number, position and sequence context. Technical
advantages
were seen when compared to DNA controls for the best LNA primer
designs,
which were typically one to a few centrally located LNA residues. LNA
advantages include increased maximum annealing temperature
ðTmaxÞ
and increased signal with limiting primer or Taq DNA polymerase.
Several
well-characterized designs exhibited different efficiencies with
different
brands of hot-start enzymes. Many shorter LNA primers were found to be
functional compared to same-length non-functional native DNA controls.
These results show that LNA-substituted PCR primers have potential for
use in difficult PCR techniques, such as multiplex amplification at
higher
Tmax; once firm LNA primer design rules are established.

We have developed a new class of
probes for homogeneous nucleic acid detection based on the proposed displacement
hybridization. Our probes consist of two complementary
oligodeoxyribonucleotides of different length labeled with a fluorophore and a quencher in
close proximity in the duplex. The probes on their own are quenched, but they become
fluorescent upon displacement hybridization with the target. These probes
display complete discrimination between a perfectly matched target and
single nucleotide mismatch targets. A comparison of double-stranded probes with
corresponding linear probes confirms that thepresence of the
complementary strand significantly enhances their specificity. Using four such probes labeled
with different color fluorophores, each designed to recognize a different target,
we have demonstrated that multiple targets can be distinguished in the same
solution, even if they differ from one another by as little as a single nucleotide.
Double-stranded probes were used in real-time nucleic acid amplifications as
either probes or as primers. In addition to its extreme specificity and
flexibility, the new class of probes is simple to design and synthesize, has low cost and
high sensitivity and is accessible to a wide range of labels. This class of
probes should find applications in a variety of areas wherever high specificity of
nucleic acid hybridization is relevant.

Simple and reliable genotyping
technology is a key to success for high-throughput genetic screening
in the post-genome era. Here we have developed a new real-time PCR genotyping
approach that uses displacement hybridization-based probes:
displacing probes. The specificity of displacing probes could be simply assessed
through denaturation analysis before genotyping was implemented, and the probes
designed with maximal specificity also showed the greatest detection
sensitivity. The ease in design, the simple single-dye labeling chemistry and the
capability to adopt degenerated negative strands for point mutation genotyping make the
displacing probes both cost effective and easy to use. The feasibility of
this method was first tested by detecting the C282Y mutation in the human
hemochromatosis gene. The robustness of this approach was then validated by
simultaneous genotyping of five different types of mutation in the human
beta-globin gene. Sixty-two human genomic DNA samples with nine known genotypes were
accurately detected, 32 random clinical samples were successfully screened and 114
double-blind DNA samples were all correctly genotyped. The combined merits of
reliability, flexibility and simplicity should make this method suitable for
routine clinical testing and large-scale genetic screening.

Vanvik et al.
developed light-up probes for sequence specific detection of nucleic
acids in homogeneous solution. The probes are made of the nucleic acid
analogue, PNA, and an assymmetric cyanine dye, which upon bind binding
to nucleic acids becomes intesively fluorescent. Under optimum
conditions the probe fluorescence increases 50-fold upon binding to
target DNA and the fluorescence can be observed by the naked eye.

Accurate
over a broad range of target concentrations—detect differences of up to
5 orders of magnitude

Bright
fluorescent signal—works on any qPCR instrument

Introducing
the BD QZyme™ Assay for quantitative PCR (qPCR), a
novel DNA amplification system for the realtime detection and
quantification of specific cDNA and genomic DNA targets. Compatible
with all real-time PCR instruments and readily adapted for use in
single or multiplex analyses, BD QZyme Assays can accurately measure
fewer than 10
copies of target DNA. The assays are easy to set up
and require no optimization since they rely on a
single set of PCR cycling parameters, which can be
universally applied for the detection of any genomic
DNA or mRNA target. The dynamic range, or ability of the assay to
accurately measure differences in target concentration, is
extraordinarily broad, typically extending over
5 orders of magnitude for high-abundance genes.

Figure:
The BD QZyme™ Assay. The 5' Primer is
comprised of a target-specific
sequence joined to the inactive (antisense) strand of the DNAzyme.
During
amplification, amplicons are produced that contain active (sense)
copies of the DNAzyme. The accumulation of amplicons is accompanied by
an increase in fluorescence, produced by the action of the DNAzyme on
its fluorogenic substrate. Elements not drawn to scale. CS = Cleavage
Site.

The Amplifluor™
Universal Detection System is a proprietary technology platform that
allows
the simultaneous amplification and detection of nucleic acids within
a closed reaction vessel. The method is based upon the incorporation
of energy transfer-labeled hairpin primers into the amplification
product.
Amplifluor™ hairpin primers are designed so that a fluorescent signal
is generated only when the primer is unfolded during its incorporation
into an amplification product. The fluorescence signal produced
directly
correlates with the accumulation of PCR product at each cycle.
Unincorporated
Amplifluor™ primers have an extremely low fluorescence signal
eliminating
the need to purify the PCR reaction prior to quantitation; therefore,
PCR and fluorescent signal detection can occur in a single reaction
vessel.
Signal is measured either during the reaction (real-time) or after the
last cycle of the reaction (endpoint). Amplifluor™ primers also perform
extremely well for in situ PCR applications using paraffin-embedded
tissues (see References utilizing Amplifluor™ Technology).

This is a new
detection system for real-time qPCR which does not require the use of a
probe. Simply
put, the LUX system is composed of two primers, just one being
label(FAM
or JOE). The quenching of the fluorescence of the labeled primer isprovided by the
secondary structure of the primer (LOOP configuration thanksto the addition of a
5' tail) and the terminal dG-dC or dC-dG base pair whenthe dye is attached within four
nucleotides from the 3´-end.

A brochure and
a manual is available on this productto enable you to
have a better idea of the capacity of our product.

- low
price: Just 1 labeled primer and a regular primer, no need
for a quencher
or a probe.- easy to design:
software freely available on Invitrogen website- multiplexing
possibility (advantage over SYBR green)- melting curve
possibility (advantage over TaqMan)

A novel real-time
quantitative polymerase chain reaction (PCR) method
using an attached universal template (UT) probe is
described. The UT is an approximately 20 base
attachment to the 5-prime end of a PCR primer, and it can
hybridize with a complementary TaqMan probe. One
of the advantages of this method is that different
target DNA sequences can be detected employing
the same UT probe, which substantially reduces the
cost of real-time PCR setup. In addition,
this method could be used for simultaneous detection using a
6-carboxy-fluoresceinlabeled UT probe for the target
gene and a 5-hexachloro- fluorescein-labeled UT
probe for the reference gene in a multiplex
reaction. Moreover, the requirement of target DNA
length for UT±PCR analysis is
relatively flexible, and it could be as short as 56 bp in this report,
suggesting the possibility of detecting target
DNA from partially degraded samples. The UT±PCR system
with degenerate primers could also be designed
to screen homologous genes. Taken together, our
results suggest that the UT±PCR
technique is efficient, reliable, inexpensive and less labor-intensive
for quantitative PCR analysis.